| Acute renal failure (AFR) is a sudden loss of kidney function which characterized as decreased glomerular filtration, elevated blood urea nitrogen (BUN) and creatinine in several days or even hours. AFR may lead to a number of complications, including the imbalance of water and electrolyte, acid-base and induce acute uremia. Renal ischemia reperfusion injury (RIRI) is one of the main causes for ischemic AFR. It occurs inevitably in organ transplant. Many factors have been identified to be involved in development of RIRI, such as free radical, calcium overload, energy metabolism dysfunction, inflammatory mediators, adhesion molecules and cytokines.If the acute kidney injury is not treated appropriately, the pathological process may continue and last for weeks and years, and finally deteriorate into chronic renal insufficiency, or chronic renal failure. In 2010, Kidney International magazine continuously published five reviews which are all focused on long-term protecting strategy of the transplanted organs. In this regard, inspection on changes in 24 hrs after renal ischemia reperfusion (I/R) or any signal time point is obviously not enough. In this project, we observed the comprehensive changes after reperfusion from 6 hr to 21 days, and investigated the pathological progress of renal injury from acute to subacute period.Hydrogen sulfide (H2S) was gradually accepted as the third gasotransmitter after nitric oxide (NO) and carbon monoxide (CO). Recent reports suggest H2S exhibits different physiological functions in the body. Most tissues produce significant amount of H2S, while the brain, cardiovascular system, liver and kidney are believe having the highest H2S generation rate. The formation of endogenous H2S was mainly from catalysis of pyridoxal 5'-phosphate (vitamin B6)-dependent by cystathionine-?-synthase (CBS), cystathionine-y-lyase (CSE) or cysteine transferase. The three enzymes have different distributions in the body. In kidney, it is reported that both CSE and CBS are highly expressed, but little was known about the physiological role of H2S in kidney.In our previous study, we observed the change of endogenous H2S in early stage of diabetes and high glucose treated renal mesangial cells. Our result demonstrated that decrease in endogenous H2S, which was due to the decrease of CSE in diabetes, was correlated to renal oxidative stress, and related to development of diabetic nephropathy. In cultured glomerular mesangial cells, NaHS, a donor of exogenous H2S, depressed the ROS generation stimulated by high glucose. ROS generation is the most important factor in ischemia reperfusion injury. It has been reported H2S ease myocardial injury from cardiac ischemia reperfusion. In the present study, we investigated the change of endogenous H2S production after renal ischemia reperfusion and its possible role in RIRI.Renal mesangial cells (MCs) is an inherent cell in renal glomeruli. It provides structural support for glomerular capillaries and participates in glomerular blood flow regulation via contract. Large numbers of researches have demonstrated that the abnormalities of mesangial cell proliferation and/or extracellular matrix (ECM) production and secretion play key role in renal glomerulosclerosis. In I/R induced renal injury study, the previous researchers mainly paid attention to renal tubular cells and their function. However, in our primary study, we noticed that there was a great increase in inflammatory cytokines and fibrosis related factors expression induced by I/R operation, and these changes were much earlier and greater in renal cortex. It suggested that glomerular cells may play a central role in I/R renal injury. However, little data discussed the function of MCs in RIRI, especially in the subacute period. In this project, hypoxia/reoxygenation (H/R) on cultured renal MCs was used to mimic I/R model in vivo to investigate the possible role of MCs in I/R renal injury.The endoplasmic reticulum (ER), an important intracellular organelle of eukaryocyte, is the factory for folding and maturation of newly synthesized transmembrane and secretory proteins. Accumulation of unfold or misfolded proteins in ER leads to ER stress (ERS) and triggers the unfolded protein response (UPR). UPR is a highly conserved self-protective mechanism of cells by which ameliorates the accumulation of unfold or misfolded proteins in the ER. However, The fate and destination of the cell are also depended on the manifestation of ER stress, if ER stress is severe or protracted, cell apoptosis may be induced.Ischemia and reperfusion activates cellular ERS, and ERS was involved in the progress or recovery of I/R injury. It has been reported that I/R induced ER stress leads to cell apoptosis and organ damage. On the other side, Xi et al. reported that ERS precondition ameliorated I/R induced renal injury. ER stress, likes a double-edged sword, can be related to target organ injury, or protection. Its function in I/R renal injury and the interaction between endogenous H2S and ERS will also be observed in this project. This paper is composed of three parts:Part I. Decrease in endogenous H2S production in kidney after I/R and administration of exogenous H2S ameliorated I/R induced renal injury1. The H2S concentration in kidneys was quantified at 60 min ischemia followed by 6 hr reperfusion. The H2S level was significantly lower in I/R renal cortex than that of the sham-operated animals. However, no significant change was observed in renal medulla. Supplement of H2S by administration of NaHS (dosage) had no significant effect on serum creatinine, BUN and morphology in the sham-operated group.2. Bilateral renal ischemia reperfusion model was used in the experiment. In this model, renal ischemia was induced by clamping renal artery for 60 min then restoring the blood perfusion. Sham-operated animals experienced the same procedure except clamping the renal artery. The tissues were harvest at 6 hr,24 hr,3 days,7 days, or 21 days after reperfusion. The observation on urine protein, serum creatinine and BUN suggested that renal injury induced by I/R appeared as early as 6 hr after surgery, and lasted until to the end of observation. HE stain of the tissue slices confirmed the pathological changes.3. In I/R+NaHS group, exogenous H2S was supplemented by intraperitoneal injection of NaHS (100?g/kg)10 min before reperfusion. The results of urine protein, serum creatinine. BUN and morphological changes suggested that exogenous H2S supplementation before reperfusion had no significant effect on acute renal injury induced by I/R, however, it ameliorated renal injury in subacute period.4. Inflammatory cytokines play important role in I/R induced injury. Interleukin 6 (IL-6) was one of the most important mediators in renal non-specific inflammation responses, and was highly expressed in kidney. In the present study, the serum IL-6 level increased more than 3 times in I/R and I/R+NaHS groups 6 hr after reperfusion. 24 hr after reperfusion, the concentration of IL-6 in serum tended to decrease. However, it was still significant higher than that in the sham-operated groups till 21 days. On the contrary, the serum IL-6 values in I/R+NaHS group did not increase obviously. What's more, in the time point of 21 days, the value in I/R+NaHS group was significantly lower than that in I/R group. I/R operation induced a significant increase in IL-6 mRNA level in renal cortex. The peak of IL-6 mRNA appeared at 24 hrs after surgery, when the level of IL-6 mRNA level in I/R group was about eight times higher than that in sham-operated group. The mRNA level was also elevated in I/R+NaHS group, but it was significantly lower than that in I/R group. The IL-6 mRNA level in renal medulla did not change significantly 6 hrs after surgery. In I/R group, IL-6 mRNA level in renal medulla increased from the time point of 24 hr. and reached the peak at 3 days. In I/R+NaHS group, it increased from the time point of 3 days, and was lower than that in I/R group. These results suggested that the elevation in IL-6 in kidney was locally original, and the change is earlier in renal cortex than in medulla. IL-6 production induced by I/R could be partially inhibited by exogenous H2S. We also observed the changes in monocyte chemoattractant protein-1 (MCP-1) mRNA, another inflammatory cytokine in renal cortex, and it showed similar changes as IL-6. The changes in both IL-6 and MCP-1 confirmed that non-specific inflammation response is induced by I/R and H2S may play an anti-inflammation function in kidney.5. Renal fibrosis is the common feature of various chronic renal diseases. Transforming growth factor-?(TGF-?) plays the key role in the progress of renal fibrosis. Real time PCR result showed, TGF-?mRNA level did not change obviously 24 hrs after surgery. From the time point of 3 days, TGF-?expression increased in both I/R and I/R+NaHS group. However, the value in I/R+NaHS group was lower than that in I/R group. Fibronectin (FN) is one of the important extracellular matrix (ECM) component participates in renal fibrosis. As a target protein regulated by TGF-?, the change in FN mRNA level in renal cortex was similar to TGF-?. These results suggested that expressions of the fibrosis element and its related factor were elevated in subacute period which might lead to the progress of renal fibrosis.Part?. H2S inhibited production of ROS induced by I/R in kidney and functional changes in cultured renal MCs induced by H/R.1. We measured superoxide levels in kidney by lucigenin-enhanced chemiluminescence assay. The production of ROS was increased significantly after I/R operation, and it was attenuated by exogenous H2S supplementation.2. Renal mesangial cells are major contributors to the ECM which play key role in glomerulosclerosis and renal fibrosis. Hypoxia/reoxygenation model of cultured renal MCs was used to investigate the effect of H/R on cell biological functions. ROS generation in the cells was quantified by fluorescent probes assay. The result showed that hypoxia for 2 hrs induced a significant increase in ROS. The ROS level returned to the control value after reoxygenation for 2 hrs. However, it increased again after that, and reached the peak value at 6 hr, then return back again to the control level at 12 hr. Administration of NaHS (30?M) either before hypoxia or at the time of reoxygenation inhibited ROS elevation induced by H/R. However. NaHS administration before hypoxia had no effect on the second elevation in ROS while NaHS administration at the time of reoxygenation inhibited the second elevation. These results suggested that H/R stimulated generation of ROS in cultured MCs and the elevation of ROS showed two peaks, the earlier peak was related to hypoxia, while the later one, which appeared at 6 hrs after reoxygenation, might be related to reoxygenation.3. IL-6 mRNA level in cultured renal MCs and IL-6 in supernatant were significantly increased after H/R. The elevations were inhibited by NaHS administration. The result suggested that H2S inhibited non-specific inflammation response mediated by IL-6. What's more, NaHS inhibited H/R induced cell proliferation. Since the proliferation and ECM production of MCs is essential to glomerulosclerosis and renal fibrosis, the effects of exogenous H2S, that inhibits on non-specific inflammation response and cell proliferation, decrease the expression of fibrosis related factors in vitro, may related to ameliorating effect of renal ischemia reperfusion injury in vivo.4. H/R induced generation of ROS was completely inhibited by diphenylenechloride iodonium (DPI), a NADPH oxidase inhibitor, suggested that the ROS over production induced by H/R was produced by NADPH oxidase. DPI administration also inhibited H/R induced IL-6 mRNA level increase and cell proliferation, which suggested that ROS elevation induced by H/R was related to IL-6 produce and cell proliferation.Part?. Renal ischemia reperfusion activated endoplasmic reticulum stress.1. We measured the GRP78 protein level, the maker protein of ERS, in the kidney of sham, I/R and I/R+NaHS groups by Western blot. GRP78 protein level elevated 24 hr after reperfusion, and returned to the baseline after 3 days. In renal MCs, H/R induced GRP78 protein increase and phosphoration of eIF2?suggested that ERS could be activated by I/R or H/R.2. Exogenous H2S inhibited I/R induced GRP78 protein increase in kidney and H/R treated MCs. as well as eIF2?phosphoration. NaHS alone had no effect on ERS in the control cells.3. ERS in renal MCs could be induced by thapsigargin (TG) or tunicamycin (TM). Pretreatment of NaHS had no significant effect on TG or TM induced GRP78 protein increase or eIF2?phosphoration. These results suggested that H2S had no direct effect on ERS. Combined with the previous result, we speculate that the inhibition of ERS by H2S administration might be a result of decreased ROS or inflammatory cytokines, however, the exact mechanism needs further investigation.In summary, the present study recorded the changing process in renal functions, morphology, local ROS, inflammatory factors and fibrosis related factors in acute and subacute periods after RIRI. It provides meaningful data for elucidation the mechanism of renal injury in early stage of I/R. In the present subject, combined analysis of the data from I/R animal model and H/R treated cells strengthen the solid of conclusion. Data from both in vivo and in vitro studies showed that ROS generation elevated after ischemia and reperfusion, meanwhile, endogenous H2S production decreased. In vivo, ROS stimulated IL-6 production and cell proliferation in MCs, which could be inhibited by H2S supplementation. Meanwhile, in vitro study, administration of H2S donor significantly attenuated I/R induced over expression of inflammatory cytokines and upregulation of TGF-?and ECM synthesis, which played key role in renal fibrosis. All these results suggested that local nonspecific inflammatory response which was induced by over production of ROS, may result in TGF-?and ECM synthesis and lead to renal fibrosis. Amelioration of I/R induced renal damage by H2S supplementation might be mediated by attenuating ROS generation, then the nonspecific inflammatory response in local renal tissues. |
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